Cellular responses to pore-forming toxins and regulation/function of the exocyst

University Of California, San Diego, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant): Cellular function depends critically on the ability of the cell to direct proteins and membranes to their correct destinations, and diseases such as cancer and polycystic kidney disease are associated with dysregulation of important trafficking mediators. One of these key trafficking mediators is the exocyst, an octameric cytoplasmic complex conserved from yeast to mammals [1]. The exocyst is best known for its proposed role as a tethering complex involved in directed vesicle fusion at the plasma membrane in a wide range of critical cellular processes. However, much is still unknown about how the exocyst is regulated and what its functions in the cell are. It is also becoming increasingly clear that the exocyst does not always act as a single unit; instead different subunits have been individually linked to different exocyst-mediated processes. Our lab recently published a genome-wide RNAi screen, performed in the Caenorhabditis elegans roundworm, that strongly implicates subunits of the exocyst as important components of the cellular program of responses to holes formed in the plasma membrane following attack by pore-forming toxin (PFT) proteins [2]. PFTs are the most common bacterial virulence factors and are secreted by several major human pathogens, including Staphylococcus aureus, Bacillus anthracis, and Vibrio cholerae [3-5]. Studies of PFTs are important not only for understanding bacterial pathogenesis but also for understanding how cells deal with membrane damage in general. Our lab has discovered that PFT defenses in C. elegans include signaling through mitogen activated protein kinase (MAPK) pathways as well as membrane trafficking events, including increased endocytosis, shedding of plasma membrane, and membrane resealing [2, 6, 7]. These same responses have also been observed in mammalian cells [8-12]. As the exocyst has also been connected to MAPK signal transduction pathways [13-15] and to membrane trafficking in other contexts [16], my aim is to use the study of PFT-induced responses in the C. elegans intestine as a new system for probing exocyst function and regulation. I will use RNAi to determine which exocyst subunits are required for the signal transduction and membrane trafficking events associated with cellular responses to PFTs. I will also construct a reporter worm expressing fluorescently-tagged exocyst subunits in order to observe exocyst activation and to subsequently determine which previously-identified PFT defense genes [2] regulate exocyst activation. Results from these studies will shed new light on the role and regulation of the exocyst and its component subunits in fundamental membrane trafficking processes and will have important implications for understanding how cells deal with membrane damage and for controlling bacterial infection and controlling of diseases whose pathogenesis involves dysregulation of exocytic trafficking.